DESCRIPTION: (provided by the applicant): Dengue viruses, serotypes 1-4 are currently the most important mosquito-borne viruses affecting humans. It is estimated that 2.5 billion humans are at risk for infection. About 50-100 million cases of dengue fever (DF) and >100,000 cases of life-threatening dengue hemorrhagic fever (DHF) occur annually worldwide. A major factor contributing to the dramatic global emergence of DF and DI-IF as public health concerns is the proliferation throughout the tropics of Aedes aegypti, the principal vector of DEN viruses. The long-term goal of this research is to define molecular strategies for reducing or eliminating the competence of Ae. aegypti to transmit DEN and other flaviviruses. Flavivirus replication in mosquito cells can be inhibited by intracellular expression of double-stranded RNA. Characteristics of RNA-mediated interference in mosquito cells are very similar to those of evolutionarily conserved defense mechanisms in plants and other invertebrates such as Drosophila melanogaster. We hypothesize that RNA-mediated interference (RMI) in mosquitoes is a robust cellular process, homologous to those in other organisms, and that it can be manipulated to eliminate mosquito vector competence for flaviviruses. In this proposal, we plan studies to determine the detailed mechanism of RMI in mosquitoes. In addition to our mechanistic studies, we will examine the role of RMI in maintenance of flavivirus persistent infections in mosquitoes and in determining vector competence for flavivirus transmission. The specific aims of the proposal are as follows: 1.To define the mechanism for RNA-mediated interference (RMI)-to flaviviruses in mosquitoes. The molecular and biochemical characteristics of RMI in cultured mosquito cells and Ac. aegypti will be compared to those of posttranscriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in Drosophila. 2:To determine the relationship between RNAi and persistent arbovirus infections in mosquitoes. The possibility that a counterdefensive dengue viral suppressor of RMI will be examined by use of a dengue virus infectious clone. 3. To determine the role of RNAi in refractoriness/susceptibility of mosquitoes to flaviviruses. Ac. aegypti genes associated with the RMI response will be characterized and mapped.